US5589168A - Probiotic - Google Patents

Probiotic Download PDF

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US5589168A
US5589168A US08/300,207 US30020794A US5589168A US 5589168 A US5589168 A US 5589168A US 30020794 A US30020794 A US 30020794A US 5589168 A US5589168 A US 5589168A
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ibs
strain
patient
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patients
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William D. Allen
Margaret A. Linggood
Philip Porter
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Givaudan Nederland Services BV
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Unilever Patent Holdings BV
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
    • A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
    • A23D7/0056—Spread compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/21—Streptococcus, lactococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales

Definitions

  • This invention relates to so-called “probiotic” organisms, and to compositions containing them, which are useful for promoting or maintaining the health and general well-being of humans and animals.
  • strain PR88 Enterococcus faecium bacterium
  • IBS irritable bowel syndrome
  • Strain PR88 confers similar benefits in cases of other severe abdominal complaints, such as so-called “inflammatory bowel disease” (IBD).
  • IBD inflammatory bowel disease
  • the beneficial properties of strain PR88 may also facilitate the re-establishment of normal gastroenteric functions in human patients who have undergone severe abdominal surgery or other treatments (such as chemotherapy or radiotherapy) which have resulted in their natural gastroenteric functions becoming unbalanced.
  • strain PR88 can advantageously be administered to animals, especially domesticated mammals such as pigs, cattle, horses and domestic pets.
  • the invention provides Entercococcus organisms which, if administered as a single oral dose of 10 11 viable organisms to a human patient diagnosed as suffering from IBS, maintain a faecal excretion rate of at least 10 5 Enterococcus organisms per gm of wet faeces from said patient for more than 5 days post-treatment, and alleviate one or more symptoms of IBS in said patient within 28 days, preferably 14 days, post-treatment.
  • Preferred organisms of the invention are capable of alleviating one or more symptoms of IBS within 10 days, more preferably within 7 days, post-treatment. Preferred organisms can also maintain the desired faecal excretion rate for more than 10 days post-treatment.
  • the invention provides Enterococcus organisms which, if administered as single daily oral doses each of 10 10 viable organisms, over a continuous period of 14 days, (preferably merely 10 days) to a human patient diagnosed as suffering from IBS, maintain a faecal excretion rate of at least 10 5 Entercococcus organisms per gm of wet faeces from said patient throughout said treatment period and alleviate one or more symptoms of IBS in said patient during said treatment period.
  • strain PR88 will be used hereinafter to include any Entercococcus strain encompassed by the general definitions given in the two immediately preceding paragraphs.
  • One embodiment of the invention resides in the novel strain PR88 itself.
  • the invention extends to mutants and derivatives of strain PR88 which exhibit similar or greater probiotic efficacy, especially in relation to IBS.
  • a further embodiment is the use of strain PR88 in the treatment of humans or animals.
  • the invention provides an edible composition for administration to a human patient exhibiting symptoms of gastroenteric disorder, comprising viable organisms of strain PR88 in a carrier material.
  • the carrier material is aqueous.
  • the carrier material should be pharmaceutically-acceptable.
  • composition will normally be packaged with instructions recommending a mode of administration to the patient, e.g. specifying dosage quantity and frequency of administration, based on clinical trials conducted with a particular formulation in accordance with the invention.
  • a further embodiment of the invention is the use of strain PR88 in the manufacture of a composition for administration to humans or animals.
  • a composition can comprise, for example, organisms of strain PR88 in a pharmaceutically acceptable carrier medium which can be fed to a patient or administered by intubation.
  • lyophilised viable PR88 organisms can be dispensed in solid form, e.g. as a capsule.
  • the invention also comprises edible products such as fermented milk products, e.g. yoghurts, containing strain PR88.
  • edible products such as fermented milk products, e.g. yoghurts, containing strain PR88.
  • Non-milk based products can also be provided, such as yoghurt-like compositions containing milk substitutes such as soya.
  • the strain can also be incorporated in the aqueous phase of edible emulsions, such as margarine, low-fat spreads, or dressings.
  • the composition will contain at least about 10 3 , preferably at least about 10 6 , and ideally from about 10 9 to about 10 10 viable PR88 organisms per gm.
  • the strain PR88 can be cultured industrially to provide commercial quantities of organisms, by growth under typical bacterial fermentation conditions.
  • the strain can be supplied commercially as a concentrate in an edible carrier (e.g. water) which does not induce growth of the bacteria.
  • an edible carrier e.g. water
  • Another alternative is a freeze dried composition containing viable PR88 organisms. Such compositions can be added to appropriate foodstuffs during manufacture of the foodstuffs, to provide probiotic products. Care should be taken that subsequent processing of the foodstuff does not lead to the loss of the desired probiotic activity.
  • Edible Capsules made for example from gelatin, containing the strain PR88 (preferably in lyophylised form) can be a useful product form for prophylactic or therapeutic use.
  • PR88 is capable of fermenting milk, it is not especially efficient in this respect. If PR88 is to be incorporated in a probiotic yoghurt, for example, it may be convenient to produce the yoghurt by means of a conventional yoghurt strain, e.g. a Lactobacillus, and to add the PR88 organisms later.
  • a conventional yoghurt strain e.g. a Lactobacillus
  • strain PR88 as a probiotic additive in human foods or animal feed.
  • strain PR88 will generally be used in the form of intact viable cells, the invention also extends to non-viable cells of strain PR88 such as killed cultures and other compositions containing beneficial factors expressed by strain PR88.
  • An edible composition comprising viable organisms of strain NCIMB 40371, packaged with instructions recommending a mode of administration to a human patient exhibiting symptoms of gastroenteric disorder;
  • a controlled diet for a human patient exhibiting symptoms of IBS including such a composition
  • IBS Irritable Bowel Syndrome
  • PGE prostagladin E2
  • Exogenous PGE exerts a potent secretory effect on the intestinal epithelium and can evoke copious watery diarrhoea in humans following oral, intrajejunal or parenteral administration.
  • PGs also affect intestinal motility.
  • PGE is synthesized locally in the small intestine and its synthesis/release is normally under hormonal and neutral control. Abnormal release of PGE would clearly give rise to adverse symptoms and individuals with IBS-diarrhoea often do have higher than normal PGE levels (Rask-Madsen & Bukhave, 1985).
  • One condition that may trigger the release of PGE is food intolerance. Jones et al (1982) reported that specific foods provoked symptoms of IBS in 14/21 patients and this was associated with increased levels of rectal PGE.
  • Probiotic therapy can benefit IBS patients, but it is necessary to identify a suitable bacterial strain.
  • the mode of action of probiotics is not at present well-understood. The following three parameters seem important.
  • the strain should be a natural intestinal strain, non-pathogenic and non-toxic.
  • a suitable, safe delivery system should be available for giving the patient large numbers of viable cells.
  • the strain should be capable of surviving and metabolizing in the gut environment, e.g. be resistant to low pH and organic acids.
  • Strain PR88 is a naturally-occurring gram-positive coccus identified as Streptococcus (Enterococcus) faecium.
  • the API 20S code of the strain is 5357 511 (very good identification Ent.faecium) and it grows on thallous acetate--tetrazolium glucose agar giving white colonies.
  • PR88 will ferment sugars such as glucose to give acids thereby lowering the pH to about 4.5 and will "yoghurt" milk media after 19-24 hours at 37° C but the yoghurts used in this study were incubated for 48 hours (a milk-intolerant patient was found to react to 24h, but not 48h, yoghurts). Counts of PR88 in the yoghurts were usually between 10 8 and 10 9 per gram and the pH was about 4.6.
  • PR88 was grown in 500 ml aliquots of GYSM* for intracaecal inoculation and 100 ml aliquots of DS* for oral inoculation.
  • HVD high volume diarrhoea ⁇
  • Table 1 The average daily faecal weights of the HVD patients were measured (over a period of 3-4 days) before and after treatment. The condition scores of all patients were assessed before and after treatment by the simple index method of Harvey and Bradshaw (1980).
  • the PR88 culture was initially administered by intracaecal intubation and their intestinal PR88 levels were then maintained by daily yoghurts. Five patients had the oral yoghurt therapy only (see Table 1). Later, each individual reduced their yoghurt consumption (one pot every second or third day) until they reached what they considered to be the right frequency to maintain improved health.
  • the faecal bacteriology of healthy individuals as measured by anaerobe/aerobe ratios can vary considerably from day to day although it is possible to calculate the ⁇ normal range ⁇ for faecal samples treated in a standard way.
  • the faecal bacteriology counts of IBS patients gave a higher percentage of abnormal anaerobe/aerobe ratios than healthy individuals, but after probiotic treatment gave ratios in the normal range.
  • the abnormal ratios could be either above or below the norm and many IBS patients gave similar ratios when normal and when in diarrhoeal breakdown. Therefore it can be inferred that although some IBS patients may have an abnormal faecal flora as measured by crude anaerobe/aerobe ratios but there is no characteristic IBS profile. (Imbalances within the aerobe or anaerobe group would not be shown up by this method).
  • PR88 yoghurts milk much more slowly than a commercial culture but it can survive and proliferate in the alimentary tract.
  • PR88 could implant and maintain large intestinal populations for several weeks.
  • yoghurts taken orally could also result in high numbers of the strain in the intestine, as patients in the trial usually had faecal levels of PR88 of 10 6 -10 9 per gram.
  • Probiotic therapy with PR88 was very effective at alleviating the symptoms of IBS, particularly diarrhoea-associated IBS.
  • the average daily faecal weights of the patients were determined before and after probiotic treatment and clearly demonstrated the efficacy of PR88.
  • the two ⁇ failures ⁇ were both intolerant of cow's milk and also reacted adversely to dairy yoghurts.
  • the three patients with intermittent diarrhoea also responded well to yoghurt treatment as did 2 individuals with IBS without diarrhoea.
  • the other symptoms of IBS such as abdominal pain, migraine-like headache and arthritic pains and swelling of the joints could also be relieved by the probiotic therapy (see Table 4)
  • yoghurts were a safe and efficient means of administering PR88. Viable counts were about 10 8 /ml so one yoghurt contained 10 10 bacteria. The yoghurt pH was about 4.5 so this reduced the risks of contamination; pathogens such as salmonella and listeria are not able to survive at low pHs. PR88 survived well in the acid yoghurt environment and the yoghurt-grown bacteria were in a suitable metabolic state to colonise the gut.
  • Administration of a daily oral dose of 1 ⁇ 10 10 of Enterococcus faecium PR88 for a period of 14 days resulted in a daily faecal excretion of strain PR88 in excess of 1 ⁇ 10 5 organisms per gram and an amelioration of the symptoms consistent with at least a partial remission.
  • Enterococcus faecium strain PR88 confers a unique benefit in terms of amelioration of the symptoms of IBS consistent with at least a partial remission, not conferred by other strains of Enterococcus faecium, whether occurring naturally or administered medicinally.
  • Enterococcus faecium strain PR88 was maintained in vials frozen in liquid nitrogen.
  • An inoculum for commercial scale production was propagated from frozen vials in a medium consisting of hydrolysed soy protein, yeast extract, glucose, and buffering salts (eg monopotassium hydrogen phosphate and disodium hydrogen phosphate) and a mineral source (eg magnesium sulphate).
  • a medium consisting of at least one nitrogen source (eg non-fat dry milk, hydrolyzed soy protein, corn steep liquor and yeast extract) is used, with a carbon and energy source (eg glucose or lactose).
  • a mineral source eg magnesium sulphate
  • Buffering salts eg. monopotassium hydrogen phosphate and disodium hydrogen phosphate
  • the ingredients are uniformly blended in water and sterilized in a pressurized fermenter vessel.
  • the vessel has aseptic attachments for monitoring and controlling temperature and pH. Immediately after exhaustion of the carbon source(s) or just before complete exhaustion, the vessel is cooled to refrigeration temperature, and the cells are harvested by either centriguation or ultrafiltration.
  • the cold cell concentrate can either be frozen after the addition of cryoprotectants such as sterile milk, lactose or sucrose solution, glycerol, or monosodium glutamate, or frozen as shallow layers in sterile trays and freeze-dried.
  • cryoprotectants such as sterile milk, lactose or sucrose solution, glycerol, or monosodium glutamate, or frozen as shallow layers in sterile trays and freeze-dried.
  • a medium consisting of non-fat dry milk at 1%, yeast extract at 1%, lactose or glucose at 2-3%, magnesium sulfate at 0.1%, and a combination of sodium phosphate and potassium phosphate at 0.9% was used.
  • the sterilized medium had an initial pH between 6.3-6.6. Incubation was at 37° C. The pH stat was set at 5.8. After 14-15 hours the cells were harvested by centrifugation to obtain 10 ⁇ concentration. A cryoprotective mixture of milk and lactose was added to the concentrate before lyophilization. A count of 1.5 ⁇ 10 11 colony forming units per gram of the cell powder was obtained.
  • Enterococcus faecium strain PR88 prepared as a cell powder in accordance with Example 5, is added by simple admixture to a conventional fermented milk product, eg yoghurt, in an amount sufficient to provide amout 10 9 viable PR88 organisms per 100 gm of product.
  • Probiotic benefits are derived by a human consuming the product on a regular basis, consistent with normal levels of consumption of such products.
  • Enterococcus faecium strain PR88 is added to a beverage product in an amount sufficient to provide about 10 9 viable PR88 organisms per 100 ml of product.
  • the beverage is essentially aqueous, but may contain protein, e.g. cadein or soy, plus minerals and electrolytes.
  • probiotic organisms of the invention will be used in medicaments, prophylactics and foodstuffs generally as essentially pure cultures.
  • Bowel-flora alteration a potential cure for inflammatory bowel disease and irritable bowel syndrome.

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US08/300,207 1991-04-08 1994-09-02 Probiotic Expired - Fee Related US5589168A (en)

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US08/300,207 US5589168A (en) 1991-04-08 1994-09-02 Probiotic
US08/697,893 US5728380A (en) 1991-04-08 1996-09-03 Priobiotic containing enterococcus faecium strain NCIMB 40371

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GB9107305 1991-04-08
GB919107305A GB9107305D0 (en) 1991-04-08 1991-04-08 Probiotic
US86499292A 1992-04-08 1992-04-08
US08/300,207 US5589168A (en) 1991-04-08 1994-09-02 Probiotic

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EP (1) EP0508701B1 (de)
JP (1) JP3186202B2 (de)
KR (1) KR0123456B1 (de)
AT (1) ATE140265T1 (de)
AU (1) AU652815B2 (de)
CA (1) CA2064954A1 (de)
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Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5879719A (en) * 1997-08-28 1999-03-09 Midwest Zoological Research, Inc. Process for control, elimination or inhibition of salmonellae in reptiles and/or amphibians
US5922317A (en) * 1996-01-04 1999-07-13 The United States Of America As Represented By The Secretary Of The Navy Accelerated gas removal from divers' tissues utilizing gas metabolizing bacteria
US5965128A (en) * 1997-08-13 1999-10-12 University Of Georgia Research Foundation Inc. Control of enterohemorrhagic E. coli 0157:H7 in cattle by probiotic bacteria and specific strains of E. coli
WO2002083926A2 (en) 2001-04-17 2002-10-24 Cedars-Sinai Medical Center Methods of diagnosing and treating small intestinal bacterial overgrowth (sibo) and sibo-related conditions
US6517832B1 (en) * 2001-08-24 2003-02-11 Jeffrey L. Marrongelle Formulations and methods for treating chronic migraine
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US5728380A (en) 1998-03-17
EP0508701B1 (de) 1996-07-10
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NZ242195A (en) 1995-04-27
EP0508701A3 (en) 1993-05-05
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JPH05252934A (ja) 1993-10-05
ES2089398T3 (es) 1996-10-01
DE69212034T2 (de) 1997-01-23
GR3021069T3 (en) 1996-12-31
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AU1473892A (en) 1992-10-15
ATE140265T1 (de) 1996-07-15
DE69212034D1 (de) 1996-08-14
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JP3186202B2 (ja) 2001-07-11

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